Diagnostic composition and a method of in-vivo determination of the function of the human pancreas

ABSTRACT

THERE ARE PROVIDED A DIAGNOSTIC COMPOSITION FOR THE DETERMINATION OF THE ACTIVITY OF PANCREATIC ENZYMES IN THE LIVING HUMAN BODY AND A METHOD FOR CARRYING OUT THIS EXAMINATION OF THE EXOCRINE PANCREAS FUNCTION.THE NEW DIAGNOSTIC COMPOSITION COMPRISES A MONOESTER OF FLUORESCIEN WITH A FATTY ACID HAVING 8 TO 16 CARBON ATOMS IN A PHARMACEUTICAL CARRIER. THIS NEW METHOD FOR EXAMINING THE EXOCRINE PANCREAS FUNCTION IS CARRIED OUT IN THAT A PREDETERMINED AMOUNT OF A FLUORESCEIN FATTY ACID MONOESTER IS INTRODUCED INTO AND PASSED THROUGH THE PASTROINTESTINAL TRACT, THEN THE URINE IS COLLECTED OR A PORTION OF BLOOD SERUM IS WITHDRAWN AND THE AMOUNT OF RELEASED FLUORESCEIN CONTAINED IN THE URINE OR THE BLOOD SERUM IS DETERMINED ANALYTICALLY.

United States Patent DIAGNOSTIC COMPOSITION AND A METHOD OF IN-VIVO DETERMINATION OF THE FUNCTION OF THE HUMAN PANCREAS .Iiirgen Meyer-Bertenrath, Hanan, Hans Kaliarmk, Marburg, Hans-Georg Rey, Mannheim-Waldhof, Gerhard Michal, Tutzing, Upper Bavaria, and Ernst-Werner Busch, Lampertheim, Germany, assignors to Boehringer Mannheim GmbH, Mannheim-Waldhof, Germany No Drawing. Filed Sept. 3, 1970, Ser. No. 69,425

Claims priority, application Germany, Sept. 10, 1969, P 19 45 663.4 Int. Cl. C0711 7/42; C09k 3/00; G01ln 31/00 US. Cl. 424-7 8 Claims ABSTRACT OF THE DISCLOSURE There are provided a diagnostic composition for the determination of the activity of pancreatic enzymes in the living human body and a method for carrying out this examination of the exocrine pancreas function. This new diagnostic composition comprises a monoester of fluorescein with a fatty acid having 8 to 16 carbon atoms in a pharmaceutical carrier. This new method for examining the exocrine pancreas function is carried out in that a predetermined amount of a fluorescein fatty acid monoester is introduced into and passed through the gastrointestinal tract, then the urine is collected or a portion of blood serum is withdrawn and the amount of released fluorescein contained in the urine or the blood serum is determined analytically.

The human abdominal salivary glands excrete a series of enzymes which are of great importance to digestion. Determination of the activity of these enzymes, which are excreted in the duodenum, gives information about the functioning of the pancreas and is important in the diagnosis of, in particular, pancreatitis, which is difficult to detect. In the diagnosis of pancreas disorders, it is important that the determination of the pancreas-enzyme concentrations in the duodenal fluid or blood serum can be carried out quickly and in the simplest way possible with good reliability, in order that chronic pancreatitis may be diagnosed.

According to the hitherto known method practiced usually, a specimen amount of duodenal fluid is sampled from the patient by means of a catheter, the sample is added to a substrate, such as triolein, and, after incubation, is examined for its content of free fatty acids. This method is extremely time-consuming, unpleasant for the patient and liable to a large number of errors.

Two of the inventors have already proposed a diagnostic method for determining pancreas enzymes in body fluids comprising a fluorescein-diester of a fatty acid, preferably a diester of lauric or myristic acid. In the journal, Klin. Wschr. 47, pages 221-223 (1969), it is described to administer colorless diesters of fluorescein with long-chain fatty acids, particularly lauric acid, orally in the form of intestine-soluble capsules, whereby the non-readsorbable and water-insoluble diestcr are enzymatically hydrolyzed in the small intestine to readsorbable fluorescein which then is eliminated by the kidneys into the urine and can be analyzed therein, e.g. photometrically or fluorimetrically. With this method which signifies great progress in pancreas diagnosis, it is necessary that, after administration of the diagnostic agent, the urine is collected for about 10 hours and analyzed, otherwise sufficient accuracy cannot always be achieved for hydrolysis of the fluorescein diester by the pancreas esterases takes place relatively slowly.

It is an object of the present invention to avoid these disadvantages and to succeed with essentially better and faster results in the diagnosis of human pancreas disorders.

A further object of the present invention is to provide a new and novel diagnostic composition comprising amonoester of fluorescein with a fatty acid containing 8 to 16 carbon atoms, preferably lauric acid, in a pharmaceutical carrier for determining pancreas enzymes in body fluids.

It is also an object of the present invention to provide a method for testing the exocrine pancreas function in the living human body, wherein a predetermined amount of a monoester of fluorescein with a fatty acid having 8 to 16 carbon atoms is passed into the gastrointestinal tract, the amount of fluorescein excreted in a defined period of time and contained in the urine or released into the blood serum is analyzed.

These and other objects and advantages will become apparent from the following description of the invention herein disclosed.

While in order to detect lipase in the serum, the monocapric acid ester of fluorescein has already been used (Fleischer and Schwartz, Abstr. Papers, 156th ACS-Meeting, 1968, Biol. 269), this assay relates to an automatic in-vitro determination, only, which does not set out the possibility of using fluorescein monoesters as a diagnostic agent for in-vivo analysis of the pancreas function.

The fluoroscein monoesters for use in this invention are colorless or slightly yellowish substances which are insoluble or only very slightly soluble in water and are not reabsorbable on its way through the human body. They are decomposed in the small intestine by the enzymes of the pancreatic secretion, in particular the esterases of pancreas, specifically into fatty acids and fluorescein. Unlike the fluorescein monoesters, the fluorescein resulting from the decomposition of the esters: is a readsorbable substance. The reabsorbed fluorescein is excreted in the urine; the concentration of fluorescein found there is a measure of the function of the pancreas.

Accordingly, the mono fatty acid esters of fluorescein are suitable as substrates for enzymes, so that in the hydrolysis by the enzyme of the pancreas secretion, the quinonoid form of the dyestuff fluorescein is released in reabsorbable form.

The following reaction takes place:

Pancreas Colorless 3- or 6- enzymes fluorescein mono fatty acid ester +1 fatty acid of fluorescein +1H20 In the serum or urine the reabsorbed fluorescein can be quantitively analyzed, photometrically or fluorimetrically or by any suitable analyzing method known in the art.

In addition, the present invention providing a fluorescein mono fatty acid ester shows a number of improvements and advantages over the art, such as:

(i) Since the molecular weight of the mono fatty acid esters of fluorescein is relatively low, the dosage units may be decreased of 25 percent without the fluorescein level in the organism decreasing.

(ii) Since there is to split only one ester bond even the control of smaller enzyme activities: becomes more sensitive.

(iii) Since the monoesters according to the invention are apparently decomposed more quickly as compared to the diesters, and following the hydrolysis are excreted in the duodenum about twice as quickly as compared to the diesters under like conditions, the collection period of the urine can be substantially shortened since the excretion of the fluorescein is finished after a few hours. Since the diesters are not decomposed in a precisely known way, but probably in two stages by different enzymes, the correlation was relatively unreliable until now. This uncertainty does not exist with the use of monoesters according to the invention which are apparently decomposed by the pancreas esterases in only one reaction stage.

The fluorescein monoesters are preferably used in the conventional preparative forms, such as tablets, pills, capsules or drops. Moreover, it is preferred to incorporate in addition the usual pharmaceutical excipients, such as fillers, sweeteners and the other usual additives. Those forms are preferred which contain the esters in a finelydivided, e.g. micronized or suspended form. The solid preparative forms, such as pills or coated tablets, can also be provided with coatings which have a retarding solubility and are soluble in the stomach or intestine. In order that the alkaline condition which prevails in the duodenum, is reliably controlled on dissolution of the diagnostic composition of the invention, it is also possible to add buffer substances, e.g. buffer salts which in solution adjust the pH value in the range of 7.5-8.5.

For oral administration of the diagnostic composition according to the invention, at a certain time, the composition is administered to the patient in one of these preparative forms in dosage units with a content of fluorescein mono fatty acid esters with 8 to 16 carbon atoms in the range of about 0.1 mmol. (about 51 mg.) to about 1.0 mmol. (about 514 mg), preferably of about 0.5 mmol. (257 mg); after certain periods of time, the fluorescein, resulting from the hydrolysis, is analyzed in the combined portions of the urine, photometrically or fluorimetrically as described in the periodical Hoppe Seylers, Z. Phys. Chem. 349, pp. 1071-1072 (1968).

The liberation of pancreas enzymes only takes place when the pancreas is stimulated by the absorption of food; it is therefore relatively simple to determine, firstly in the fasting state, the mostly negligible basic readsorption value of fluorescein and compare this control value with the test value resulting after the pancreas having been excited, for example after a specimen breakfast. The value thus obtained stands in direct relation to the enzymatic activity of the pancreas. In the case of a deficiency in the pancreas, reduced fluorescein amounts are detected in the blood serum and urine compared to the normal value.

Since reabsorption and liberation disorders, such as changing partial liberation via the gall bladder, can under certain circumstances, impair the quantitative detection of the amounts of fluorescein released by the pancreas enzymes, it can be of advantage to fix the 100% value for each patient by a delayed administration of an amount of unesterified fluorescein which is equimolar with the test dose and to relate the fluorescein liberation reached by the enzyme activity thereto. In order to determine this individual standard, another dyestulf With similar liberation properties may obviously be used in place of fluorescein. In this case, it is possible to administer the diagnostic agent, the fluorescein mono ester, together with the dyestufi" other than fluorescein which is used.

tA further method according to the present invention is the n-vivo determination in the blood serum which is carried out as follows: Each 60 min., 90 min., and 120 min., resp. after the oral administration of a test capsule of the diagnostic agent according to the invention containing 0.5 mmol. of a fluorescein monoester, portions of blood serum are withdrawn from the patient. The portions are collected and the collected portions of the blood serum are diluted with a 0.1 N aqueous sodium hydroxide solution, 1n the ratio of about 1:5 (or, with low values of fluorescein, in the ratio of about 1:2). The diluted solutions are incubated for minutes at 90 C. and are then cooled down and centrifugated. The supernatant liquid is measured in a photometer at the wavelength of 492 m or fluorimetrically analyzed. Since the blood serum has a specific inherent color, a correction must be made with a zerocontrol figure obtained from measuring of a specimen of blood serum (which is free of fluorescein).

The fluorescein mono esters used according to the invention are produced in that fluorescein is reacted in a molar proportion of 1:1 with a reactive derivative of a fatty acid containing 8 to 16 carbon atoms in an inert solvent, preferably in chloroform, if required under heating. The reaction can be promoted by the addition of tertiary or aromatic amines, such as for example triethylamine 0r pyridine. Acid chlorides or imidazolides may be used as the reactive derivatives. The fluorescein mono acld ester indicated in the following examples has a melting point of 109-1l1 C.

The following examples are given only to explain the invention in further detail and are not intended to restrict it in any way.

EXAMPLE 1 A healthy test patient in the fasting state received orally a test sample containing 257 mg. (0.5 mmol.) of fluorescein mono lauric acid ester together with 700 ml. tea. The urine was then collected for 6 hours and examined for its content of fluorescein.

Since under these conditions practically no pancreas enzymes are excreted, only a negligible amount of fluorescein was located in the urine examined.

If free fluorescein (0.5 mmol.) is employed under the same conditions, it is recovered in small amounts of 30- 50% in the collected urine.

EXAMPLE 2 A healthy test person received, together with as test breakfast consisting of 50 g. white bread, 20 g. butter and 700 ml. tea, which serves to stimulate the pancreas, 257 mg. (0.5 mmol.) of fluorescein mono lauric acid ester. The urine was then collected for 6 hours and examined for its content of fluorescein.

It was seen that the pancreas was stimulated of the individual standard being extracted via the kidneys.

In the event of difiicult pancreopathic conditions, this value dropped to 10% of the individual standard.

Although the invention has been described in considerable detail in the foregoing, it is to be understood that such detail is solely for the purpose of illustration and that many variations can be made by those skilled in the art without departing from the spirit and scope of the invention except as set forth in the claims.

What is claimed is: I

1. A diagnostic composition for determining the activity of pancreas enzymes in the living human body, comprising an oral dosage unit pharmaceutical carrier containing a predetermined oral dosage unit amount of about 51 mg. to about 514 mg. of a monoester of fluorescein with fatty acids having 8 to 16 carbon atoms.

2. A diagnostic composition according to claim 1, comprising a fluorescein 3- or 6-monolauric acid ester.

3. A diagnostic composition according to claim 1, characterized in that said fluorescein monoester or mixtures thereof is in micronized form.

4. A diagnostic composition according to claim 1, comprising in addition to said fluorescein monoester a buffering agent adjusting the pH value to 7.5-8.5.

5. A diagnostic composition according to claim 1, characterized in that said fluorescein monoester is incorporated in a pill, coated tablet or a capsule having a coating which has a retarding solubility and is soluble in the stomach or dissolves in the alkaline medium of the small intestine.

6. A method for examining the exocrine pancreas function in the living human body, characterized in that a predetermined oral dosage unit amount of about 51 mg. to about 514 mg. of a mono fatty acid ester of fluorescein is orally ingested and thereby is passed into the gastrointestinal tract and the released fluorescein found in the serum or excreted with the urine is analyzed in vitro photometrically or fluorimetrically.

7. A method according to claim 6, characterized in that the pharmaceutical preparative form consists of a pill, a coated tablet or a capsule with a coating which only dis- References Cited UNITED STATES PATENTS 2,801,203 7/1957 Leb et a1 424-35 X 3,341,417 9/ 1967 Sinaiko 424-2 2,996,431 8/ 1961 Barry 4247 2,770,571 11/1956 Vance et a1 20684 OTHER REFERENCES Schwartz et al., Advances in Clinical Chemistry, 13, 113, 126, 127, 128, 129, 130, 154, 155, 157 (1970),

Diagnostic Biochemical Methods in Pancreatic Disease.

Fleisher et al., An Automated Procedure for the Pluorometric Determination of Lipase and Esterase Activity, Paper, BioL, 269, Abstracts of Papers, 15 6th National Meeting, American Chem. 800., Atlantic City, N.J., Sept. 8-13 (1968).

Chem. Abs., 47578a (Sept. 15, 1969), 3703415 (Sept. 1, 1969).

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SHEP K. ROSE, Primary Examiner US. Cl. X.R. 

